Retroviral transfer of herpes simplex virus thymidine kinase and beta-galactosidase genes into U937 cells with bicistronic vector

被引:9
作者
DiIanni, M
Casciari, C
Ciurnelli, R
Fulvi, A
Bagnis, C
Sadelain, M
Lucheroni, F
Mannoni, P
Stella, CC
Martelli, MF
Tabilio, A
机构
[1] UNIV PERUGIA,DEPT CLIN MED PATHOL & PHARMACOL,HEMATOL & CLIN IMMUNOL SECT,MONTELUCE POLICLIN,I-06100 PERUGIA,ITALY
[2] INST J PAOLI I CALMETTES,DEPT GENE THERAPY,F-13009 MARSEILLE,FRANCE
[3] MEM SLOAN KETTERING CANC CTR,DEPT HUMAN GENET,NEW YORK,NY 10021
[4] UNIV PARMA,CHAIR HEMATOL,I-43100 PARMA,ITALY
[5] UNIV PARMA,BONE MARROW TRANSPLANTAT UNIT,I-43100 PARMA,ITALY
关键词
herpes simplex virus thymidine kinase gene; bacterial beta-galactosidase gene; bicistronic vector; retroviral transfer; U937; IRES sequence;
D O I
10.1016/S0145-2126(97)00074-X
中图分类号
R73 [肿瘤学];
学科分类号
100214 ;
摘要
In this study we describe a new retroviral vector utilizing an internal ribosome entry site (IRES) from encephalomyocarditis virus to co-express two genes. One is the herpes simplex virus type 1 thymidine kinase gene (HSV-TK) which induces sensitivity to ganciclovir, and the second is the bacterial beta-galactosidase gene (LacZ) which was revealed by an histochemical staining with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal), We enginereed the U937 human cell line to co-express both genes and monitored transduced cells using X-Gal staining. Several transduced clones were selected. The clones exhibiting X-Gal positive cells were sensitive to ganciclovir treatment (1 mu g/ml) while X-Gal negative clones were not. Monoclonal cell lines showed a single copy of the provirus integrated in their genome with the TK-IRES-LacZ sequence stably inserted in all clones. The band distribution pattern of the proviral DNA differed only at the long terminal repeat (LTR) level, Northern blot analysis of an X-Gal positive/ganciclovir sensitive clone showed an mRNA band of 6 kb with both LacZ and TK probes, An X-Gal negative/ganciclovir resistant clone was negative with both probes. This report shows: (1) a therapeutic gene can be linked to a marker gene by an IRES element achieving equivalent expression of both proteins; (2) the co-expression of a marker gene makes fluorescein-di-beta-D-galactopyranoside staining possible, and consequently separation of cells expressing the LacZ gene by fluorescence activated cell sorting, Thus the cells expressing the HSV-Tk gene are enriched; (3) the use of a marker gene such as LacZ could open up interesting perspectives in gene therapy protocols because of the opportunity to monitor the transduced cells using a simple cytochemical stain. (C) 1997 Elsevier Science Ltd.
引用
收藏
页码:951 / 959
页数:9
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